Identification of an entomopathogenic bacterium, Serratia sp. ANU101, and its hemolytic activity.

Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.

Identification of two entomopathogenic bacteria from a nematode pathogenic to the Oriental beetle, Blitopertha orientalis.

A pathogenic nematode, Butlerius sp., was isolated from Oriental beetle, Blitopertha orientalis. The infective juveniles exhibited dose- as well as time-dependent entomopathogenicity on the larvae of B. orientalis. Two bacterial species, Providencia vermicola (KACC 91278) and Flavobacterium sp. (KACC 91279), were isolated from the infective juveniles and identified. P. vermicola outnumbered Flavobacterium sp. in the nematode host, in which the colony density of P. vermicola was found to be 21 times higher than that of Flavobacterium sp. However, when the two bacterial species were cocultured in culture media without the nematode host, they showed similar growth rates. Both bacteria induced significant entomopathogenicity against Spodoptera exigua larvae infesting economically important vegetable crops, where P. vermicola was more potent than Flavobacterium sp.

Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria.

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae. In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics. The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase. The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum. The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities. Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one). With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound. This study reports a new antibiotic compound synthesized by X. nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.